title: BubR1 is required for the mitotic block induced by combretastatin-A4 and a novel cis-restricted ß-lactam analogue in human cancer cells.
abstract: BubR1 is a well-defined guardian of the mitotic spindle, initiating mitotic arrest in response to the lack of tension and/or chromosome alignment across the mitotic plate. However, the role of BubR1 in combretastatin-induced cell death remains unknown. In this study, we describe the effects of combretastatin A-4 (CA-4) and a synthetic cis-restricted 3,4-diaryl-2-azetidinone (ß-lactam) analogue (CA-432) on the modulation and phosphorylation of BubR1 in human cervical cancer-derived cells. We demonstrate that CA-4 and CA-432 depolymerise the microtubular network of human cervical carcinoma-derived cells. Both compounds induced the disassembly of the microtubules and the loss of microtubule tension led to the early phosphorylation of BubR1 and the late cleavage of BubR1. The phosphorylation of BubR1 correlated with the onset of G2M cell cycle arrest whilst the cleavage of BubR1 coincided with apoptosis induced by the combretastatins. The combretastatin-induced apoptosis and the BubR1 cleavage were caspase-dependent. In vitro enzyme digests demonstrated that combretastatin-activated BubR1 is a substrate for caspase-3. Gene silencing of BubR1 with small interfering RNA severely compromised combretastatin-induced G2M cell cycle arrest with a corresponding increase in the formation of polyploid cells in both cervical and breast cancer-derived cells. In summary, BubR1 is required to maintain the G2M arrest and limit the formation of polyploid cells in response to continued combretastatin exposure. Moreover, substitution of the ethylene bridge with 3,4-diaryl-2-azetidinone did not alter the tubulin depolymerising properties or the subsequent mitotic spindle checkpoint response to CA-4 in human cancer cells.
authors.affiliation: School of Biochemistry and Immunology, Trinity College Dublin, College Green, Dublin 2, Ireland. greeneli@tcd.ie
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Greene
authors.forename: Miriam
authors.initials: M
authors.lastname: Carr
authors.forename: Niall O
authors.initials: NO
authors.lastname: Keeley
authors.forename: Mark
authors.initials: M
authors.lastname: Lawler
authors.forename: Mary J
authors.initials: MJ
authors.lastname: Meegan
authors.forename: Daniela M
authors.initials: DM
authors.lastname: Zisterer
mesh_terms: D000972:Antineoplastic Agents, Phytogenic / Q000138:chemical synthesis / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001384:Azetidines / Q000138:chemical synthesis / Q000494:pharmacology*; D053148:Caspase 3 / Q000737:chemistry / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D006139:Guaiacol / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000494:pharmacology; D006801:Humans; D008938:Mitosis / Q000187:drug effects*; D010766:Phosphorylation; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D055503:Protein Multimerization; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D013267:Stilbenes / Q000138:chemical synthesis / Q000494:pharmacology*; D014404:Tubulin / Q000378:metabolism
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D001384:Azetidines; C553484:CA 432; D013267:Stilbenes; D014404:Tubulin; D006139:Guaiacol; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; C584277:BUB1 protein, human; C090464:Bub1 spindle checkpoint protein; D017346:Protein Serine-Threonine Kinases; D053148:Caspase 3; C058728:fosbretabulin